Second messenger c-di-AMP regulates multiple antibiotic sensitivity pathways in Mycobacterium smegmatis by discrete mechanisms

One of the debilitating causes of high mortality in the case of tuberculosis and other bacterial infections is the resistance development against standard drugs. There are limited studies so far to describe how a bacterial second messenger molecule can directly participate in distinctive antibiotic tolerance characteristics of a cell in a mechanism-dependent manner. Here we show that intracellular cyclic di-AMP (c-di-AMP) concentration can modulate drug sensitivity of Mycobacterium smegmatis by directly interacting with either a protein effector or with the 5-UTR regions in mRNA of the genes and thus causing transcriptional downregulation of important genes in the pathways. We studied four antibiotics with different mechanisms of action: rifampicin, ciprofloxacin, erythromycin, and tobramycin and subsequently found that the level of drug sensitivity of the bacteria is directly proportional to the c-di-AMP concentration inside the cell. Further, we unraveled the underlying molecular mechanisms to delineate the specific genes and pathways regulated by c-di-AMP and hence result in differential drug sensitivity in M. smegmatis. Thus, our findings of c-di-AMP messenger controlling drug resistance phenotypes of mycobacteria against four different classes of antibiotics is a unique observation that will contribute to scientific advancement in the field. One Sentence SummaryHere we describe how second messenger c-di-AMP modulates the antibiotic sensitivity and resistance profile of M. smegmatis by diverse mechanisms.